WO2001075159A2 - Microechantillons de genes regulateurs - Google Patents

Microechantillons de genes regulateurs Download PDF

Info

Publication number
WO2001075159A2
WO2001075159A2 PCT/US2001/010055 US0110055W WO0175159A2 WO 2001075159 A2 WO2001075159 A2 WO 2001075159A2 US 0110055 W US0110055 W US 0110055W WO 0175159 A2 WO0175159 A2 WO 0175159A2
Authority
WO
WIPO (PCT)
Prior art keywords
genes
expression
box
array
primers
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2001/010055
Other languages
English (en)
Other versions
WO2001075159A3 (fr
Inventor
Eugenia Wang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jewish General Hospital
Original Assignee
Jewish General Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jewish General Hospital filed Critical Jewish General Hospital
Priority to AU2001251086A priority Critical patent/AU2001251086A1/en
Publication of WO2001075159A2 publication Critical patent/WO2001075159A2/fr
Anticipated expiration legal-status Critical
Publication of WO2001075159A3 publication Critical patent/WO2001075159A3/fr
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof

Definitions

  • microarray biochip technology is certainly the method of choice to solve the problem of complexity, and the previously impossible task of defining a genetic signature for a unique person in a cohort with accuracy and speed that are impossible by the conventional diagnostic approach. Therefore, from bench-side researchers to bedside physicians, there is intense interest in the technology of microarray analysis, for screening or identifying tens or hundreds of genes related to disease or normal states of a given person or biological system. cDNA and oligonucleotide microarrays are becoming an increasingly powerful technique for investigating gene expression patterns. In spite of the fast progress in this field, some limitations of the technique persist. One of the major obstacles is the requirement for a large amount of mRNA. Another problem with existing microarray systems is data mining; while information on expression of tens of thousands genes is absolutely vital to estimate the functions of new genes, in some instances a researcher is interested in the expression profile of only a subset of genes, in many physiological conditions.
  • Microarray technology is a fast-growing field of biomedical research, aiming to investigate changes in molecular features of hundreds of genes.
  • the multiple parallel processing of information generated from matrices of huge numbers of loci on a solid substrate has allowed the gathering of gene signatures defining specific biological states.
  • a new approach has been developed to facilitate this process wherein genes of the same regulatory modality are selected.
  • the transcriptional regulation of these genes is related to the same control element, the E-box, defined by the sequence CACGT.
  • PCR products of selected regions of all known genes either binding to this sequence or whose expression is dependent on this binding, as well as genes interacting with E-box-binding genes and control genes, are arrayed on a nylon membrane or other appropriate microchip susbstrate, which is then used as an E-box- specific microarray.
  • the transcriptionally regulated profile of E-box-related genes specific to a given cultured cell sample is then determined by unique labeled cDNAs probes produced from RNAs isolated from the culture of interest.
  • E-box binding-related genes represent a specific group of basic helix-loop-helix/leucine zipper transcription factors, recognizing the core-binding site CACGTG. They play important roles in regulation of basic cellular functions, like proliferation and apoptosis (c- Myc) or tissue-specific differentiation (Myod).
  • c- Myc proliferation and apoptosis
  • Myod tissue-specific differentiation
  • Figure 1 depicts cDNA microarray hybridization for evaluation of E- box binding-related gene expression.
  • the matrix position, with each gene's abbreviation, is written underneath each locus of three repeats of dots with identical amounts deposited; the X-coordinates denote the number 1, 2, 3, 4, and 5 positions, and Y-coordinates denote the "a" through "o" positions.
  • the matrix location for each gene triplet is then identified as X,Y coordinates. For example, 5k denotes the position of N-Myc, and 3d denotes of the position of Mad. The same coordinates are also included in Table 2.
  • Figures 2 A and 2B shows the expression profiles of E-box binding- related gene expressions in Hela cells.
  • Figure 2A -total RNA was labeled with digoxigenin in RT reaction with gene specific primers;
  • Figure 2B - mRNA was labeled with digoxigenin in RT reaction with oligo(dT) primers.
  • Arrows within the matrix show positions of: I- Hela DNA (positive control); II- lambda DNA (negative control); 777-UBC; 77-RPL-13A; F- MBP-1; K7- HPRT1. The distance between dots can be measured by the bar of 1mm.
  • Figure 3 depicts hybridization of products of multiplex PCR with 5 pair of primers with cDNA microarray. Arrows within the matrix point to: I— Mrdb; II- c-Myc p64; III- TFII-1 ; TV- ODC1 ; V- cdc25A; VI- Hela genomic DNA.
  • Figure 4 shows the relationship between concentrations of 5 genes including Mrdb, c-Myc, TFII-1, ODC1, and cdc25a, and intensity of hybridization signals. Logarithmic approximation is shown. Dot intensity is represented by the arbitrary units on the Y-axis; concentration is measured as ng/ml on the X-axis.
  • Figures 5 A and 5B show the expression profiles of E-box-related genes in Hela cells (Figure 5 A), and normal human lymphocytes (Figure 5). Arrows within the matrix show positions of: I- Aldolase C; 77- Mad4; 771- MBP-1.
  • Figures 6 A. 6B and 6C are pairwise comparisons of E-box gene expression in Hela cells and human lymphocytes. Two independent hybridizations are averaged for each type of cell.
  • Figure 6 A Three- dimensional, and Figure 6B - two-dimensional, representations of differences in gene expression. Each panel corresponds to one column in Figure 5 A, and each bar represents an individual gene.
  • E-box binding-related genes represent a specific group of basic helix-loop-helix/leucine zipper transcription factors, recognizing the core-binding site CACGTG.
  • E-box genes refer to all genes having the E-box in their promoter region, as well as E- box binding and interacting genes. They play important roles in regulation of basic cellular functions, like proliferation and apoptosis (c-Myc) or tissue- specific differentiation (Myod).
  • E-box-binding genes The best-known representative of E-box-binding genes is c-Myc, whose transactivating activity plays crucial roles in the regulation of cell cycle, proliferation and apoptosis (Eilers, Mol. Cells 9, 1-6 (1999); Dang, CN. Mol. Cell Biol. 19, 1-11 (1999); Facchini and Perm, FASEB J. 12, 633-651 (1998)). For this reason, genes interacting with or regulating expression for c-Myc, as well as some target genes whose expression is E-box-binding-dependent, are included in this microarray. Representative E-box genes are shown in Table 2.
  • Housekeeping genes are used to normalize results of expression. These are genes that are selected based on the relatively invariable levels of expression in the system which is being examined, for example, the state such as age or a particular disease. Representative housekeeping genes are shown in Table 2. These include tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, hypoxanthine phosphoribosyltransferase I (Lesh- ⁇ yhan syndrome), Major histocompatibility complex, class I, C, Ubiquitin C, Glyceraldehyde-3 -phosphate dehydrogenase, Human mR ⁇ A fragment encoding cytoplasmic actin, 60S Ribosomal protein LI 3 A, and Aldolase C.
  • a set of primers for use in detecting changes in expression of genes include the E-box regulatory sequence, are between 480 and 700 base pairs length, have a melting point between 75 and 85°C, and include non-consensus sequence with protein coding sequence, so that there is no detectable hybridization between homologous genes, more preferably where there is no hybridization between homologous genes.
  • homologous genes examples include c-myc and c-myc associated genes.
  • E-box regulatory genes described herein can be used to assess changes associated with a particular state or disease.
  • the association ofcertain E-box genes such as c-myc with cancer and neurodegeneration, and its role in apoptosis, are well established.
  • Other genes include yyl, myc-Ll, and myc-L2, which affect cells, cell components, and specific molecules, for example, cardiomysin, myotube, osteoblasts, and osteoclasts.
  • Changes in expression of individual genes can be used to assess changes in states such as age or diseases associated with cancer of tissues such as breast, prostate, and colon, immunological changes such as inflammation, neurodegenerative diseases, cardiovascular disorders, and musculoskeletal disorders, including disorders and diseases of bones such as osteoarthritis and osteoporosis, and muscle degeneration.
  • Screening The arrays can be tested by screening with labeled probes to determine if there is expression of a particular gene in the array and how much, to thereby construct a "fingerprint" of the disease or disorder at that time, using genes present in cells or tissues obtained from one or more individuals having the disease or disorder or characterized by a particular state, such as age.
  • the effect of a compound or composition on the disorder or disease or state can also be assessed by comparing the fingerprint obtained with control cells or tissues, and cells or tissues treated with the compound or obtained from an animal treated with the compound (or compounds, or dosage regime, or exposed to particular conditions). This is especially useful for initial screening of the effect of potential drugs, either to determine potential efficacy and/or toxicity. Those compounds which appear promising can then be further screened to determine if they can reduce or reverse the severity of the disease or disorder.
  • Compounds to be screened can be proteins or peptides, sugars or polysaccharides, nucleic acid molecules, or synthetic molecules.
  • a turnkey microarrayer can be purchased, with an enclosure for temperature, humidity and air quality control; a system such as the GeneMachinesTM OmniGrid (San Carlos, CA) would be sufficient.
  • a microarrayer can be built in the laboratory.
  • the Brown Laboratory web site for example, gives full details for component specifications, mechanical drawings for machined parts, a list of vendors, an assembly guide, and free microarrayer software. Operation of the tips, XYZ motion control, and computer program
  • the robotic gantry of a typical printing tip microarryer is composed of 3 individual assemblies of linear robotic tables, and motors driven by 3 corresponding amplifiers which are coupled to a motion controller in the driving computer. All of this forms the appropriate 3 -axis motion control system (i.e.: X, Y and Z axes) for microarraying.
  • the three perpendicular axes allow for sampling, printing and washing with the components of the microarryer system.
  • poly-L- lysine-coated glass slides seem to work best to immobilize the printed DNA.
  • Nylon hybridization membranes can also be used as the printing substrate, and allow for a much easier immobilization protocol, as well as better visualization if a colorimetric method is used for hybridization detection.
  • conical 96- well microplates work well by localizing small volumes of sample in the wells. When printing many different samples, 384-well microplates are best due to their higher capacity and low storage volume and the smaller sample sizes ( ⁇ 10 ⁇ l) can be used readily.
  • sample plates should be covered with an adhesive- backed plastic seal, to prevent sample loss by evaporation.
  • Samples prepared for printing are loaded into 384-well microplates, 10 ⁇ l aliquots per well. These samples can be used for up to 8 to 10 printing runs, with proper storage.
  • the printing tip delivery volume is approximately 1 nl per spot with a spot diameter of approximately 100 ⁇ m. Therefore, depending upon the surface area of the substrate being used as the chip and the number of tips used for printing, several large arrays are possible with close spacing (less than 100 um) for up to 100 chips per run. For typical experiments in this laboratory, arrays are printed in duplicate 20x20 arrays per chip with a spot spacing of 250 ⁇ m using between 20 to 30 chips per run.
  • the microtiter plates are sealed with adhesive-backed plastic covers in addition to the microplate lids. Furthermore, before using the stored samples again, the microplates are centrifuged to gather any condensate in the wells, and to localize the sample fluids at the bottom of each well.
  • Array Analyzer/Imaging system Depending upon the selected approach to hybridization analysis of the printed microarrays, a system fitted onto an existing microscope, a microarray scanner or confocal laser scanner may be purchased, or a confocal laser scanner may be built.
  • the system used to compile the digital microarray images is built around an Olympus BH-2 upright light microscope, fitted with a Sony color CCD camera, an Applied Scientific Instrumentation (Eugene, OR) X-Y scanning stage, and a fiber optic ring illuminator from Edmund Scientific Co. (Barrington, NJ).
  • EMPIX Imaging, Inc. (Mississauga, ON) assembled the system for compiling microarray images, containing a 24 bit frame grabber; it is installed in a 450 MHz P3 PC equipped with 512 Mb RAM and a 19" SVGA monitor, where the image acquisition and system control are governed under the Windows 98 operating system by Northern EclipseTM imaging software.
  • a 3COMTM 10/100 Base TX network card installed in the computer links the imaging computer to a small LAN (Lynksys, Irvine, CA), containing a color laser printer and two other computers used for image analysis and data storage.
  • the size of the arrays and individual spots dictates the use of low power objectives (either 2.5X or 4X) and the X-Y scanning stage to capture the image of the entire array.
  • Probes are labeled with DIG-dUTP in a reverse transcription reaction; target/probe hybridization is detected with anti-DIG-coupled alkaline phosphatase, and a subsequent reaction of the alkaline phosphatase with an NBT/BCIP stain/substrate.
  • This method requires the ring illuminator to distinguish artifacts from array spots on the stained hybridization membranes. Otherwise, if poly-L-lysine coated glass slides are used as the microarray printing substrate, illumination of the microarray specimen is carried out normally.
  • the compiled montage can be transferred by way of the network to the computer stations devoted to image analysis and data storage.
  • the microarray images are created as TIFF files; before quantitation can begin, the raw digital images are filtered to bear only the microarray signal data, aligned in Adobe PhotoShopTM software, and then transferred to the GeneAnalyzer microarray analysis software.
  • GeneAnalyzer removes the background, and the reduced digital microarray images are passed through an image location routine to optimally localize the spots of the microarray image.
  • the GeneAnalyzer software has "grabbed" the individual spots of the reduced digital microarray image, the program can proceed to quantitate the density of the individual spots.
  • Each spot on the microarray is then regarded as an individual signal, and its intensity serves as the foundation of the data needed to reflect the hybridization reaction. After comparison with appropriate positive and negative controls for nonspecific reactions, true signal value is subtracted from noise to produce the desired information on each hybridization reaction.
  • microarray spot density data are transferred into an analysis routine in the mathematical analysis software, MATLAB, for graphical representation of all data; the density values, as well as the respective calculated values, of all digitized microarray data are tabulated in a Microsoft ExcelTM spreadsheet. A full record of the progression of images, tabulated data and all graphical representations can immediately be printed to complete the microarray experiment analysis. Labels for Probes and Detection
  • Microarrays typically contain at separate sites nanomolar (less than picogram) quantities of individual genes, cDNAs, or ESTs on a substrate such as a nitrocellulose or silicon plate, or photolithographically prepared glass substrate.
  • the arrays are hybridized to cDNA probes using standard techniques with gene-specific primer mixes.
  • the nucleic acid to be analyzed — the target — is isolated, amplified and labeled, typically with a fluorescent reporter group, radiolabel or phosphorous label probe.
  • the array is inserted into the scanner, where patterns of hybridization are detected.
  • the hybridization data are collected as light emitted from the reporter groups already incorporated into the target, which is now bound to the probe array. Probes that perfectly match the target generally produce stronger signals than those that have mismatches. Since the sequence and position of each probe on the array are known, by complementarity, the identity of the target nucleic acid applied to the probe array can be determined.
  • cDNAs and ESTs can be detected by autoradiography or phosphorimaging ( 32 P). Fluorescent dyes are also used, and are commercially available from suppliers such as Clontech.
  • the label is digoxigenin (DIG). This specific enzymatic labeling probe allows the end result of detecting hybridization reaction intensity by colorimetric evaluation of alkaline phosphatase-coupled antibody to DIG.
  • DIG digoxigenin
  • Example 1 Digoxigenin Enzymatic Detection for Microarray Analysis of E-Box Binding Related Gene Expression.
  • E-box-binding proteins as well as c-Myc-regulating, -interacting and target genes, were chosen from different data bases - GeneAtlas (http://www.citi2.fr/GENATLAS), GeneCards (http://bioinfo.weizmann.ac.il/cards), GenBank
  • a pair of primers was generated with the help of Primer3 software (Rosen and Skaletsky (1998) Primer3. Code available at http://www-genome.wi.mit.edu/ genome software/other/primer3.htm .
  • the program parameters were chosen in such a way that the melting temperature of the amplicon should be close to 80°C but not more than 88°C or less than 75°C, the length of the amplicon was to be generally around 450 bp (with a few outliers between 300 and 700 bp), with primer annealing temperature about 60°C, and average length of primers 23 bp.
  • RNA and DNA were isolated from approximately 10 8 HeLa cell cultures and human peripheral lymphocytes isolated from fresh blood aliquots using Trizol reagent (Gibco BRL, Burlington, ON). DNA and RNA concentrations and quality were determined by spectrophotometric and gel electrophoresis analysis in 0.8 or 2% agarose gels, respectively.
  • Poly(A) + RNA was isolated from 150 ⁇ g of total RNA using the Oligotex mRNA kit (Qiagen, Mississauga, ON), according to the manufacturer's instructions. Amplification and purification of probes
  • RNA 10 ⁇ g was reverse-transcribed in 40 ⁇ l reaction, using 200 U of MMLV (Gibco BRL, Burlington, ON) according to the manufacturer's instructions.
  • Two PCR reactions for each pair of primers were conducted in a total volume of 100 ⁇ l, in a GeneAmp PCR system 9700 (PE Applied Biosystems, Norwalk, CT).
  • Each 50 ⁇ l reaction (10 mM Tris-HCl, ⁇ H8.6, 50 mM KC1, 0.1% Triton X-100, 1.5 mM MgCl 2 , 0.5 mM of each dNTP, 20 pM of each primer, 1.25 U of Taq DNA polymerase (Amersham Pharmacia Biotech, Baie d'Urfe, QC) and 10 ⁇ l of RT reaction or 100 ng of genomic DNA) was thermal-cycled as follows: first cycle at 94°C for 5 min, 35 cycles at 94°C for 45 sec, at 60°C for 1 min and at 72°C for 30 sec, the last cycle at 72°C for 7 min.
  • Probes that could not be amplified in RT-PCR were extracted from genomic DNA, with the condition that the primers were selected in the 3' region of a gene. Size and yield of PCR products were determined by gel electrophoresis in 2% agarose. Then PCR products were purified from solution or agarose gel bands, following preparative agarose gel electrophoresis (if byproducts were determined), using GFX columns (Amersham Pharmacia Biotech, Baie d'Urfe, QC). After purification, concentrations of all probes were estimated by agarose gel electrophoresis, and adjusted to approximately lOO ng/ ⁇ l. Robotic arraying
  • PCR products in 2x standard salt solution were arrayed in triplicates from 384-well plates, utilizing a GeneMachinesTM OmniGrid microarrayer (Genomic Instrumentation Services, San Carlos, CA) equipped with ChipMaker2 tips (Telechem International, San Jose, CA). The spacing between dots was 400 ⁇ m. The positions of genes in this array are indicated in Table 4.
  • Microarrays were printed on Hybond-N or Hybond-N+ nylon membranes (Amersham Pharmacia Biotech, Baie d'Urfe, QC), attached to standard glass slides with tape. Before and after each 10 slides with membranes, regular slides were inserted to inspect printing quality.
  • membranes were UN irradiated at 50 mJ (GS Gene linker, Bio-Rad, Hercules, CA) to immobilize the D ⁇ A; then fragments of membranes containing arrays (approximately 1 x 1.5 cm) were cut off, denaturated in boiling water for 5 min, rinsed in 0.1% SDS for 5 min, and used for prehybridization. After the UN irradiation step, membranes can be stored attached to glass slides.
  • GS Gene linker Bio-Rad, Hercules, CA
  • DIG-labeled cD ⁇ A for hybridization
  • GSP gene-specific primers
  • 1 nM of each primer that was used in RT-PCR reactions to prepare probes was mixed in a total volume of 250 ⁇ l.
  • Digoxigenin (DIG)-labeled targets were produced in RT reaction as follows: 1 ⁇ l of GSP, 4 ⁇ g of total R ⁇ A, and R ⁇ Ase-free water in total volume of 14 ⁇ l were heated at 65°C for 15 min to denature the R ⁇ A, and then kept at room temperature for 5 min for primer annealing. Alternatively, 2 ⁇ g of mR ⁇ A and 400 ng of oligo(dT) 1 .
  • the reaction mix containing 8 ⁇ l of 5x first strand buffer supplied by the enzyme's manufacturer, 2 ⁇ l of 10 mM mix of d ATP, dCTP and dGTP (final concentration 500 ⁇ M each), 4 ⁇ l of 0.1 M DDT, 0.7 ⁇ l RNAguard, 31 U/ ⁇ l (Amersham Pharmacia Biotech, Baie d'Urfe, QC), 10 ⁇ l of a 2 mM mix of 19:1 dTTP:DIG-l 1-dUTP (Roche, Laval, QC) and 2 ⁇ l (200 U/ ⁇ l) of Moloney murine leukemia virus reverse transcriptase (MMLV RT) (Gibco BRL, Burlington, ON), was added.
  • MMLV RT Moloney murine leukemia virus reverse transcriptase
  • Reaction was carried out at 37°C for 1 h, followed by enzyme degradation at 94°C for 5 min in GeneAmp 9700.
  • Omniscript reverse transcriptase (Qiagen, Mississauga, ON) was used according to the manufacturer's instructions. Labeling reactions were purified on GFX columns; this step eliminates all labeled products shorter than 100 bp, as well as unincorporated nucleotides, primers and protein.
  • DIG Easy Hyb buffer (Roche, Laval, QC), or formamide buffer containing 50% deionized formamide, 5x SSC, 2% blocking solution (Roche, Laval, QC), 0.1% ⁇ -lauroylsarcosine, 0.02% SDS, 100 ⁇ g/ml denaturated salmon D ⁇ A, were used.
  • Membranes were pre-hybridized at 42°C for 2 h in a hybridization oven (Autoblot, Bellco, Nineland, ⁇ J). Hybridization was performed at 42°C overnight in 1 ml or less of hybridization solution, in 5-ml Falcon tubes. The concentration of labeled probes in the hybridization mix constituted 10 ng/ml. Before hybridization the probes were denaturated at 65 C for 10 min in hybridization solution.
  • hybridization membranes were rinsed (unless mentioned specially) twice with lxSSC, 0.1% SDS for 15 min at room temperature, and then withprewarmed O.lxSSC, 0.1% SDS for 15 min at 68°C.
  • membranes were rinsed in more stringent conditions, i.e. twice in 2xSSC, 0.1% SDS at 68°C. for 30 min, and twice in O.lxSSC, 0.1% SDS at 68°C for 30 min.
  • membranes were blocked for 1.5 h in 1% blocking solution under slight agitation, and then treated for 30 min in 10 ml of alkaline phosphatase-conjugated sheep anti-digoxigenin antibody (Roche, Laval, QC), diluted 1:1000 for colorimetric staining, or 1:10000 for chemiluminescent detection.
  • membranes were rinsed three times for 15 min in rinsing buffer, equilibrated for 2 min in detection buffer (0.1 M Tris-HCl, 0.15 M ⁇ aCl, pH 9.5), and stained with 175 ⁇ g/ml 5-Bromo-4-chloro-3-indolyl-phosphate, toluidine salt (BCIP), and 330 ⁇ g/ml ⁇ itro blue tetrazolium chloride ( ⁇ BT) in detection buffer.
  • detection buffer 0.1 M Tris-HCl, 0.15 M ⁇ aCl, pH 9.5
  • detection buffer 0.1 M Tris-HCl, 0.15 M ⁇ aCl, pH 9.5
  • detection buffer 0.1 M Tris-HCl, 0.15 M ⁇ aCl, pH 9.5
  • detection buffer 0.1 M Tris-HCl, 0.15 M ⁇ aCl, pH 9.5
  • ⁇ BT toluidine salt
  • 1:100 dilution of CSPD was applied, and chemiluminescence
  • Arrays were scanned on an Olympus microscope equipped with a Multiscan-4 System (Applied Scientific Instrumentation, Eugene, OR) and a color CCD Sony 950 camera. Data acquisition and montage of different fields of view into one file were accomplished with the help of the Northern Eclipse Imaging System (EMPIX Imaging, Missisauga, ON). Quantitative measurements of intensity of enzymatic reaction at each dot, background subtraction, normalization to housekeeping genes, and comparison of paired hybridizations were all performed with an in-house software program. Results
  • genes were selected for arraying, including 9 housekeeping genes.
  • This set of genes contains 38 E- box binding genes, together with the Myc (c-, N-, LI and L2) family, 5 c-Myc regulating factors (ZFP161, nn ⁇ 23-H2S, MBP-1, RBMS 1 and RBMS2), 5 c- Myc interacting genes (YY1, TFII-1, PAM, MM-1 and alpha-tubulin), and 4 c- Myc target genes (prothymosin alpha, MRDB, ODC1, and cdc25A).
  • Positive controls include 9 housekeeping genes with different levels of expression
  • the average annealing temperature of primers was 60.1 ⁇ 0.9°C, which allowed all PCR reactions to be in the 96- well format. Sizes and melting temperatures of products, and annealing temperatures of primers, are represented in Table 4. The average size of PCR products for arraying, and their melting temperature, were 441 ⁇ 58 bp and 80 ⁇ 3°C, respectively. Selecting these parameters allowed hybridization and post-hybridization rinsing in stringent conditions, decreasing drastically the possibility of cross-hybridization and background level.
  • Scrupulous selection of primers could be used to distinguish in some cases between very close members of gene families (for example, USF1 and 2, ID2, 3 and 4, members of the Myc family, and so on), or between two different transcripts of c-Myc.
  • genes for example, USF1 and 2, ID2, 3 and 4, members of the Myc family, and so on
  • c-Myc there are several different transcription forms of c-Myc, transcribed from different promoters, with varying regulation properties (Bodescot and Brison Gene 174, 115-120 (1996)).
  • Selecting primers in the 1 st exon and the 2 nd -3 rd exons allowed discrimination between full-size and truncated forms of c-Myc.
  • MMLV Moloney murine leukemia virus
  • OmniScript OmniScript
  • This level of sensitivity allows detection of mRNAs of intermediate abundance, each representing more than 0.04% of total cell mRNA. Taking into account this detection level, it is estimated that for hybridization with a microarray containing about 70 genes of intermediate abundance, 7 ng of labeled probe produced from gene-specific primers should suffice. For the next hybridizations, a concentration of labeled probes of 10 ng/ml was selected. The yield of standard reverse transcription labeling reaction with gene specific primers is about 20-40 ng; therefore, one labeling reaction yields enough product for 2-4 independent hybridization reactions. In contrast to unstable radioactive probes, DIG-labeled probes can be stored and reused several times.
  • E-box genes were determined in replicating, HeLa cells and normal human lymphocytes. In lymphocytes, the most prominent alteration consisted of more than 2-fold up-regulation of E-box- related genes TCF4, MAD4 and Aldolase C. Alternatively, down-regulation of c-Myc-regulating genes MBP1 and Nm23-H2S, and small down-regulation of c-Myc and up-regulation of N-Myc, were registered in lymphocytes in comparison with HeLa cells. Expression of some c-Myc interacting and target genes was down- (MM-1, ODC1) or up-regulated (PAM, MrDb) in lymphocytes.
  • genes in the E-box microarray are all in the same category of abundance (intermediate or low abundant). Excluding highly abundant genes eliminates the problem of merging of strong signals. Merged signals in some circumstances substantially complicate the process of scanning, and create unreliable results during the data acquisition step.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La technologie des microéchantillons est un domaine de la recherche biomédicale en plein essor, dont le but est d'étudier des changements survenant dans les caractéristiques moléculaires de centaines de gènes. Le traitement parallèle multiple des informations générées par des matrices d'un nombre immense de loci sur un substrat solide a permis de réunir des signatures génétiques définissant des états biologiques spécifiques. Afin de faciliter ce processus, une nouvelle approche a été développée, qui consiste à sélectionner des gènes ayant la même modalité de régulation. La régulation transcriptionnelle de ces gènes est liée au même élément de régulation, la E-box, définie par la séquence CACGTG. Des produits PCR de régions choisies de tous les gènes connus qui se lient à cette séquence ou dont l'expression dépend de cette liaison, ainsi que des gènes interagissant avec des gènes se liant à l'E-box et des gènes de régulation, sont ordonnés sur une membrane de nylon ou à un autre substrat approprié de micropuce, qui est ensuite utilisé comme microéchantillon spécifique de l'E-box. Le profil à transcription régulée des gènes liés à l'E-box spécifiques d'un échantillon cellulaire cultivé est ensuite déterminé par des sondes ADNc étiquetées uniques obtenues à partir d'ARN isolé de la culture d'intérêt.
PCT/US2001/010055 2000-03-31 2001-03-29 Microechantillons de genes regulateurs Ceased WO2001075159A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001251086A AU2001251086A1 (en) 2000-03-31 2001-03-29 Microchip arrays of regulatory genes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US19388800P 2000-03-31 2000-03-31
US60/193,888 2000-03-31

Publications (2)

Publication Number Publication Date
WO2001075159A2 true WO2001075159A2 (fr) 2001-10-11
WO2001075159A3 WO2001075159A3 (fr) 2003-01-16

Family

ID=22715426

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2001/010055 Ceased WO2001075159A2 (fr) 2000-03-31 2001-03-29 Microechantillons de genes regulateurs
PCT/US2001/010096 Ceased WO2001075162A2 (fr) 2000-03-31 2001-03-29 Jeux ordonnes de microechantillons permettant de cribler des genes regulateurs

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/US2001/010096 Ceased WO2001075162A2 (fr) 2000-03-31 2001-03-29 Jeux ordonnes de microechantillons permettant de cribler des genes regulateurs

Country Status (3)

Country Link
US (3) US7157227B2 (fr)
AU (2) AU2001249600A1 (fr)
WO (2) WO2001075159A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004091637A3 (fr) * 2003-04-07 2004-11-25 Neostem Inc Systeme de traitement et de definition de diverses maladies au moyen de cellules souches

Families Citing this family (36)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080183395A1 (en) * 1999-06-28 2008-07-31 Michael Bevilacqua Gene expression profiling for identification, monitoring and treatment of multiple sclerosis
US6960439B2 (en) 1999-06-28 2005-11-01 Source Precision Medicine, Inc. Identification, monitoring and treatment of disease and characterization of biological condition using gene expression profiles
US6692916B2 (en) 1999-06-28 2004-02-17 Source Precision Medicine, Inc. Systems and methods for characterizing a biological condition or agent using precision gene expression profiles
US20060115826A1 (en) * 1999-06-28 2006-06-01 Michael Bevilacqua Gene expression profiling for identification monitoring and treatment of multiple sclerosis
US20030099973A1 (en) * 2001-07-18 2003-05-29 University Of Louisville Research Foundation, Inc. E-GeneChip online web service for data mining bioinformatics
US20050064446A1 (en) * 2001-11-01 2005-03-24 Minerva Biotechnologies Corporation Detection of binding species with colloidal and non-colloidal structures
US6964850B2 (en) * 2001-11-09 2005-11-15 Source Precision Medicine, Inc. Identification, monitoring and treatment of disease and characterization of biological condition using gene expression profiles
ES2374311T3 (es) * 2002-03-13 2012-02-15 Genomic Health, Inc. Obtención de perfil de expresión génica en tejidos tumorales biopsiados.
WO2003081201A2 (fr) * 2002-03-21 2003-10-02 Yissum Research Development Company Of The Hebrew University Of Jerusalem Marqueurs de cellules sanguines peripheriques utiles pour le diagnostic de la sclerose en plaques et procedes et trousses comprenant de tels marqueurs
US20040072274A1 (en) * 2002-05-09 2004-04-15 Lebrun Stewart J. System and method for visualization and digital analysis of protein and other macromolecule microarrays
WO2004046386A1 (fr) 2002-11-15 2004-06-03 Genomic Health, Inc. Etablissement de profils d'expressions genetique du cancer a recepteur de facteur de croissance epidermique positif
US20040231909A1 (en) * 2003-01-15 2004-11-25 Tai-Yang Luh Motorized vehicle having forward and backward differential structure
AU2004211955B2 (en) * 2003-02-06 2009-05-14 Cedars-Sinai Medical Center Gene expression markers for response to EGFR inhibitor drugs
WO2004074518A1 (fr) * 2003-02-20 2004-09-02 Genomic Health, Inc. Utilisation d'arn intronique pour mesurer l'expression genique
US6970240B2 (en) * 2003-03-10 2005-11-29 Applera Corporation Combination reader
AU2004248120B2 (en) * 2003-05-28 2009-04-23 Genomic Health, Inc. Gene expression markers for predicting response to chemotherapy
AU2004248140A1 (en) * 2003-05-30 2004-12-23 Cedars-Sinai Medical Center Gene expression markers for response to EGFR inhibitor drugs
DE10324912A1 (de) * 2003-05-30 2005-01-05 Siemens Ag Verfahren zur Detektion von DNA-Punktmutationen (SNP-Analyse) sowie zugehörige Anordnung
HUE050365T2 (hu) 2003-06-24 2022-09-28 Genomic Health Inc Rákkiújulás valószínûségének elõrejelzése
AU2004258085B2 (en) * 2003-07-10 2010-05-27 Genomic Health, Inc. Expression profile algorithm and test for cancer prognosis
CA2542656A1 (fr) * 2003-10-16 2005-05-06 Genomic Health, Inc. Systeme d'essai qrt-pcr pour le profilage d'expression genetique
AU2004309396B2 (en) 2003-12-23 2010-05-13 Genomic Health, Inc. Universal amplification of fragmented RNA
ES2636470T3 (es) * 2004-04-09 2017-10-05 Genomic Health, Inc. Marcadores de expresión génica para predecir la respuesta a la quimioterapia
US8173367B2 (en) * 2004-10-18 2012-05-08 Sherri Boucher In situ dilution of external controls for use in microarrays
CA2585571C (fr) * 2004-11-05 2020-01-21 Genomic Health, Inc. Prediction de reaction a la chimiotherapie au moyen de marqueurs d'expression genique
JP4939425B2 (ja) 2004-11-05 2012-05-23 ジェノミック ヘルス， インコーポレイテッド 乳癌の治療反応の予後および予測の分子指標
US20070072175A1 (en) * 2005-05-13 2007-03-29 Biogen Idec Ma Inc. Nucleotide array containing polynucleotide probes complementary to, or fragments of, cynomolgus monkey genes and the use thereof
WO2006132982A2 (fr) * 2005-06-03 2006-12-14 Aviaradx, Inc. Genes de normalisation
AU2008256851A1 (en) * 2007-05-23 2008-12-04 Oregon Health & Science University Microarray systems and methods for identifying DNA-binding proteins
EP2666873B1 (fr) 2007-07-12 2016-03-09 The Brigham and Women's Hospital, Inc. Compositions et méthodes permettant de diagnostiquer et d'évaluer des myopathies inflammatoires
JP5175569B2 (ja) * 2008-02-07 2013-04-03 ルネサスエレクトロニクス株式会社 同期電動機の駆動システム
JP2013545439A (ja) 2010-09-17 2013-12-26 プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ 多能性幹細胞の有用性および安全性の特徴決定を行うための機能的ゲノミクスアッセイ
US11085067B2 (en) 2013-06-10 2021-08-10 President And Fellows Of Harvard College Early developmental genomic assay for characterizing pluripotent stem cell utility and safety
WO2019147663A1 (fr) * 2018-01-24 2019-08-01 Freenome Holdings, Inc. Procédés et systèmes de détection d'anomalie dans les motifs d'acides nucléiques
US20230056396A1 (en) * 2020-01-17 2023-02-23 Asklepios Biopharmaceutical, Inc. Systems and methods for synthetic regulatory sequence design or production
WO2025038925A1 (fr) * 2023-08-17 2025-02-20 The Buck Institute For Aging Research Procédés de simulation de vieillissement inflammatoire, de dysfonctionnement cardiaque, de dysfonctionnement neuronal et de changements associés à un vol spatial dans des cellules et des organoïdes, et procédés d'identification et d'utilisation de composés utiles pour le traitement de changements cellulaires associés au vieillissement inflammatoire, à un dysfonctionnement cardiaque, à un dysfonctionnement neuronal et à un vol spatial

Family Cites Families (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5474796A (en) * 1991-09-04 1995-12-12 Protogene Laboratories, Inc. Method and apparatus for conducting an array of chemical reactions on a support surface
US5605662A (en) * 1993-11-01 1997-02-25 Nanogen, Inc. Active programmable electronic devices for molecular biological analysis and diagnostics
US5753431A (en) * 1993-10-13 1998-05-19 Northeastern Ohio University Cholesterol 7 α-hydroxdylase gene regulatory elements and transcription factors
US6015880A (en) * 1994-03-16 2000-01-18 California Institute Of Technology Method and substrate for performing multiple sequential reactions on a matrix
US6444463B1 (en) * 1994-05-06 2002-09-03 Fred Hutchinson Cancer Research Center Neurogenic differentiation gene neurod3 and methods for inducing differentiation of cells
US5807522A (en) * 1994-06-17 1998-09-15 The Board Of Trustees Of The Leland Stanford Junior University Methods for fabricating microarrays of biological samples
WO1998012354A1 (fr) * 1996-09-19 1998-03-26 Affymetrix, Inc. Identification de signatures de sequences moleculaires et procedes afferents
US6013437A (en) 1996-11-12 2000-01-11 Qbi Enterprises, Ltd. Method for identifying translationally regulated genes
EP0942969A1 (fr) * 1996-11-12 1999-09-22 Qbi Enterprises Ltd. Procede d'identification des genes regules par traduction
US6028189A (en) * 1997-03-20 2000-02-22 University Of Washington Solvent for oligonucleotide synthesis and methods of use
US5880261A (en) * 1997-04-03 1999-03-09 Waeber; Gerard Transcription factor Islet-Brain 1 (IB1)
JP2002504812A (ja) * 1997-05-21 2002-02-12 クローンテック ラボラトリーズ，インコーポレイテッド 核酸アレイ
US6444421B1 (en) * 1997-11-19 2002-09-03 The United States Of America As Represented By The Department Of Health And Human Services Methods for detecting intermolecular interactions in vivo and in vitro
DE69822206T2 (de) * 1997-12-19 2005-02-17 Affymetrix, Inc., Santa Clara Erkenntnisse der genomforschung für die suche nach neuartigen wirkstoffen
US6168915B1 (en) * 1998-04-24 2001-01-02 Diagnostic Hybrids, Inc. Mixed cell diagnostic systems
US6136541A (en) 1999-02-22 2000-10-24 Vialogy Corporation Method and apparatus for analyzing hybridized biochip patterns using resonance interactions employing quantum expressor functions
WO2000060126A2 (fr) * 1999-04-08 2000-10-12 Sir Mortimer B. Davis Jewish General Hospital Bioanalyse quantitative d'expression de genes dans des microechantillons
US20020124273A1 (en) * 2000-10-17 2002-09-05 Jean-Marc Roch Protein-protein interactions in neurodegenerative diseases
US20050054597A1 (en) * 2003-03-28 2005-03-10 Neo-Morgan Laboratory Incorporated Method and system for rapidly conferring a desired trait to an organism

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004091637A3 (fr) * 2003-04-07 2004-11-25 Neostem Inc Systeme de traitement et de definition de diverses maladies au moyen de cellules souches

Also Published As

Publication number Publication date
WO2001075159A3 (fr) 2003-01-16
AU2001249600A1 (en) 2001-10-15
US20020009736A1 (en) 2002-01-24
WO2001075162A3 (fr) 2003-01-16
US6759197B2 (en) 2004-07-06
US20020012932A1 (en) 2002-01-31
AU2001251086A1 (en) 2001-10-15
US20040265886A1 (en) 2004-12-30
US7157227B2 (en) 2007-01-02
WO2001075162A2 (fr) 2001-10-11

Similar Documents

Publication Publication Date Title
US6759197B2 (en) Microchip arrays of regulatory genes
Freeman et al. Fundamentals of DNA hybridization arrays for gene expression analysis
Kuhn et al. A novel, high-performance random array platform for quantitative gene expression profiling
Barrett et al. Microarrays: the use of oligonucleotides and cDNA for the analysis of gene expression
Duggan et al. Expression profiling using cDNA microarrays
US6500620B2 (en) Methods for amplifying and detecting multiple polynucleotides on a solid phase support
Shuber et al. High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes
US7897750B2 (en) Strategies for gene expression analysis
Dufva Introduction to microarray technology
Burgess Gene expression studies using microarrays
Bilitewski DNA microarrays: an introduction to the technology
US6316608B1 (en) Combined polynucleotide sequence as discrete assay endpoints
US9416419B2 (en) Methods for PCR and HLA typing using unpurified samples
Cortese The array of today
AU4224400A (en) Quantitative assay for expression of genes in microarray
Sarwal et al. Genomic analysis of renal allograft dysfunction using cDNA microarrays
Gomase et al. Microarray: an approach for current drug targets
Vo-Dinh et al. Micro arrays and biochips: Applications and potential in genomics and proteomics
Nees et al. Microarrays: spotlight on gene function and pharmacogenomics
US7371516B1 (en) Methods for determining the specificity and sensitivity of oligonucleo tides for hybridization
JP2004502129A5 (fr)
Vo-Dinh Biochips and microarrays: tools for the new medicine
Wilson et al. Experimental design and analysis of microarray data
JP3786204B2 (ja) マイクロアレイによるハイスループットスクリーニングシステム
CA2367413A1 (fr) Bioanalyse quantitative d'expression de genes dans des microechantillons

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP